Mechanisms of Urodele Limb Regeneration

This review explores the historical and current state of our knowledge about urodele limb regeneration. Topics discussed are (1) blastema formation by the proteolytic histolysis of limb tissues to release resident stem cells and mononucleate cells that undergo dedifferentiation, cell cycle entry and accumulation under the apical epidermal cap. (2) The origin, phenotypic memory, and positional memory of blastema cells. (3) The role played by macrophages in the early events of regeneration. (4) The role of neural and AEC factors and interaction between blastema cells in mitosis and distalization. (5) Models of pattern formation based on the results of axial reversal experiments, experiments on the regeneration of half and double half limbs, and experiments using retinoic acid to alter positional identity of blastema cells. (6) Possible mechanisms of distalization during normal and intercalary regeneration. (7) Is pattern formation is a self-organizing property of the blastema or dictated by chemical signals from adjacent tissues? (8) What is the future for regenerating a human limb

Center for Regenerative Biology and Medicine

poster abstractThe Indiana University Center for Regenerative Biology and Medicine (CRBM) was established at IUPUI in 2001 through a grant from the State of Indiana 21st Century Fund. In 2007, the CRBM was selected as an IUPUI Signature Center. Participating faculty come from the Schools of Science, Informatics, Medicine, and Dentistry. The Center is administered from the Department of Biology in the School of Science. Center administration consists of a Director, a Scientific Board to advise on scientific direction, and a Commercial Board to advise on technology transfer. For a complete description of the Center and its activities, see our website at www.regen.iupui.edu. Missions • Conduct multidisciplinary research aimed at understanding the mechanisms of natural regeneration, and translate the findings into regenerative therapies for tissues that fail to regenerate. • Provide graduate level academic training in regenerative biology and medicine. • Promote technology transfer. External Funding Sources CRBM faculty currently hold over $10M in research funding from a variety of Federal agencies and foundations: • NIH • NSF • NASA • W.M. Keck Foundation • Morton Cure Paralysis Foundation • American Health Assistance Foundation • American Cancer Society • Army Research Office

Regenerative Biology: New Tissues For Old

Throughout the human life cycle, tissues are regenerated either continuously to maintain tissue integrity in the face of normal cell turnover or in response to acute or chronic damage due to trauma or disease states. Blood, epithelia of skin and tubular organs, hair and nails, and bone marrow are examples of human tissues which regenerate continuously as well as in response to damage. Bone, muscle, adrenal cortex and kidney epithelium also regenerate in response to damage, and bone is continually remodeled in response to stress vectors. The response of many other vital tissues to damage, however, is not regeneration but repair by the formation of collagenous scar tissue. Scar tissue interrupts the normal tissue architecture, compromising· its function to varying degrees, depending on the severity of the injury or disease process. Some examples of diseases and injuries that cause serious impairment by scarring are third degree bums, spinal cord injuries, diabetes, macular degeneration of the neural retina and myocardial infarction. These injuries and diseases are costly in terms of healthcare dollars and potential decline in quality of life. Thus, a major goal of biomedical science is to be able to restore the structure and function of damaged or dysfunctional tissues that do not regenerate naturally. Three major approaches to tissue restoration have been developed: bionics, solid organ transplantation, and, more recently, regenerative biology, which includes cell transplantation, bioartificial tissue constructs and regrowth of new tissues from injured residual tissues in vivo. The purpose of this paper is to discuss these approaches with a particular emphasis on regenerative biology

Nerves and Proliferation of Progenitor Cells in Limb Regeneration

Nerves, in conjunction with the apical epidermal cap (AEC), play an important role in the proliferation of the mesenchymal progenitor cells comprising the blastema of regenerating urodele amphibian limbs. Reinnervation after amputation requires factors supplied by the forming blastema, and neurotrophic factors must be present at or above a quantitative threshold for mitosis of the blastema cells. The AEC forms independently of nerves, but requires nerves to be maintained. Urodele limb buds are independent of nerves for regeneration, but innervation imposes a regenerative requirement for nerve factors on their cells as they differentiate. There are three main ideas on the functional relationship between nerves, AEC, and blastema cells: (1) nerves and AEC produce factors with different roles in maintaining progenitor status and mitosis; (2) the AEC produces the factors that promote blastema cell mitosis, but requires nerves to express them; (3) blastema cells, nerves, and AEC all produce the same factor(s) that additively attain the required threshold for mitosis

Axolotl Xenografts Improve Regeneration of Xenopus Hind Limbs

poster abstractAxolotls regenerate perfect copies of amputated limbs, whereas Xenopus froglet limbs regenerate only a spike of cartilage. We asked whether axolotl muscle and cartilage xenografted from normal or GFP-labeled limbs to amputated froglet limbs, with or without treatment with cyclosporin A (CSA) and/or retinoic acid (RA), would improve Xenopus limb regeneration via the release of regeneration-promoting factors into the host limb tissue. The grafted froglet limbs were allowed to regenerate for three months to two years. We detected initial symptoms of graft vs. host disease with or without CSA treatment that subsequently disappeared. The grafted limbs first formed a spike that subsequently grew wider at the tip and after three months began to separate into 2-5 digit-like structures that continued to grow. CSA and low-dose RA treatment decreased the time at which digit formation could be detected but were not necessary for digit formation. The digit pattern was not asymmetric, thus individual digits were not identifiable. Immature muscle was detected in the regenerated limbs by trichrome and MF-20 antibody staining, and nerve fibers were detected by Luxol Fast Blue staining. In one limb with a GFP graft, a few axolotl cells were detected around the base of the digits that may have stimulated digit separation. Although the mechanism of digit formation remains obscure, we conclude that factors released by degraded axolotl tissue or surviving axolotl cells can stimulate complex tissue regeneration and initiate the first step of digital anterior-posterior pattern formation in regenerating Xenopus hind limbs. These results have significance for the possibility of stimulating the regeneration of complex mammalian structures that have been injured by trauma or disease

Stage-dependent effects of retinoic acid on regenerating urodele limbs

Following amputation through the distal zeugopodium, regenerating limbs of larvalAmbystoma mexicanum and pre and post-metamorphic Pleurodeles waltlii were treated with 150 μg of retinoic acid (RA) per gram of body weight, at the dedifferentiation, early bud, medium bud, late bud or early redifferentiation stages of regeneration. The effect of RA on regenerate morphogenesis differed as a function of the stage at which it was administered. When given during dedifferentiation or at early bud stages, RA evoked proximodistal duplications of stump segments in the regenerates. The maximum duplication index (DI) in Abystoma was achieved when RA was injected at 4 days post-amputation, which corresponds to the stage of dedifferentiation; and inPleurodeles at 10 days post-amputation, which corresponds to a stage midway between early bud and medium bud. When RA was administered at later stages, the DI declined progressively to zero or nearly zero by the stage of early redifferentiation in both species. The decline in DI was due to a decreased frequency of duplication, not to a decrease in the magnitude of duplication in individual regenerates. At the same time, there was an increase in hypomorphism and aberrant morphogenesis of both duplicating and non-duplicating regenerates. These results indicate that regenerative cells are differentially sensitive to RA in a stage-dependent way

Network based transcription factor analysis of regenerating axolotl limbs

<p>Abstract</p> <p>Background</p> <p>Studies on amphibian limb regeneration began in the early 1700's but we still do not completely understand the cellular and molecular events of this unique process. Understanding a complex biological process such as limb regeneration is more complicated than the knowledge of the individual genes or proteins involved. Here we followed a systems biology approach in an effort to construct the networks and pathways of protein interactions involved in formation of the accumulation blastema in regenerating axolotl limbs.</p> <p>Results</p> <p>We used the human orthologs of proteins previously identified by our research team as bait to identify the transcription factor (TF) pathways and networks that regulate blastema formation in amputated axolotl limbs. The five most connected factors, c-Myc, SP1, HNF4A, ESR1 and p53 regulate ~50% of the proteins in our data. Among these, c-Myc and SP1 regulate 36.2% of the proteins. c-Myc was the most highly connected TF (71 targets). Network analysis showed that TGF-β1 and fibronectin (FN) lead to the activation of these TFs. We found that other TFs known to be involved in epigenetic reprogramming, such as Klf4, Oct4, and Lin28 are also connected to c-Myc and SP1.</p> <p>Conclusions</p> <p>Our study provides a systems biology approach to how different molecular entities inter-connect with each other during the formation of an accumulation blastema in regenerating axolotl limbs. This approach provides an in silico methodology to identify proteins that are not detected by experimental methods such as proteomics but are potentially important to blastema formation. We found that the TFs, c-Myc and SP1 and their target genes could potentially play a central role in limb regeneration. Systems biology has the potential to map out numerous other pathways that are crucial to blastema formation in regeneration-competent limbs, to compare these to the pathways that characterize regeneration-deficient limbs and finally, to identify stem cell markers in regeneration.</p

Proteomic analysis of blastema formation in regenerating axolotl limbs

BACKGROUND: Following amputation, urodele salamander limbs reprogram somatic cells to form a blastema that self-organizes into the missing limb parts to restore the structure and function of the limb. To help understand the molecular basis of blastema formation, we used quantitative label-free liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS)-based methods to analyze changes in the proteome that occurred 1, 4 and 7 days post amputation (dpa) through the mid-tibia/fibula of axolotl hind limbs. RESULTS: We identified 309 unique proteins with significant fold change relative to controls (0 dpa), representing 10 biological process categories: (1) signaling, (2) Ca2+ binding and translocation, (3) transcription, (4) translation, (5) cytoskeleton, (6) extracellular matrix (ECM), (7) metabolism, (8) cell protection, (9) degradation, and (10) cell cycle. In all, 43 proteins exhibited exceptionally high fold changes. Of these, the ecotropic viral integrative factor 5 (EVI5), a cell cycle-related oncoprotein that prevents cells from entering the mitotic phase of the cell cycle prematurely, was of special interest because its fold change was exceptionally high throughout blastema formation. CONCLUSION: Our data were consistent with previous studies indicating the importance of inositol triphosphate and Ca2+ signaling in initiating the ECM and cytoskeletal remodeling characteristic of histolysis and cell dedifferentiation. In addition, the data suggested that blastema formation requires several mechanisms to avoid apoptosis, including reduced metabolism, differential regulation of proapoptotic and antiapoptotic proteins, and initiation of an unfolded protein response (UPR). Since there is virtually no mitosis during blastema formation, we propose that high levels of EVI5 function to arrest dedifferentiated cells somewhere in the G1/S/G2 phases of the cell cycle until they have accumulated under the wound epidermis and enter mitosis in response to neural and epidermal factors. Our findings indicate the general value of quantitative proteomic analysis in understanding the regeneration of complex structures